There is an almost complete absence of information on the use of the amastigote stage of Trypanosoma cruzi as source of antigenic material for immunization against the parasite and for use in immunodiagnostic assays. We propose to conduct research which will provide such information. To examine the induction of resistance, preparations of amastigotes, killed by chemical means, lysed by sonication, or inactivated by using ultraviolet light, will be used to immunize mice. Preparations of the different stage of T. cruzi, the epimastigote, similarly obtained as for the amastigotes will be used for comparison of results. Antigens of the amastigote stage will be isolated and their usefulness for immunodiagnostic assays and for immunization examined. Isolation of the amastigote antigens will be performed by affinity chromatography with lectins of different specificities and with monoclonal antibodies. A number of monoclonal antibodies that react with antigens present in the amastigote stage only are available in our laboratory. The capacity of the isolated antigens for use in immunoassays for diagnosis of Chagas' disease will be evaluated by employing immunodiagnostic techniques. Particular emphasis will be given to enzyme immunoassays and to the agglutination test with antigen-adsorbed latex particles. These assays can be improved considerably by using purified antigen preparations. The type of the immune response elicited by the antigens isolated from the amastigotes will be determined by techniques devised to measure both cell-mediated and antibody responses. The capacity of the isolated antigens to induce resistance against infection with T. cruzi will be examined by immunization of animals with the purified antigens alone and in combination with organisms or compounds that act as adjuvants and are known to enhance the immune responses. The proposed research will provide significant and needed data on the amastigote stage of T. cruzi which participates actively in the pathogenesis of the infection with the parasite but has not received adequate attention from scientists working with T. cruzi and Chagas' disease.